Hsp60

Het-1A (150,000) cells were seeded in triplicate 35mm tissue culture treated dishes (Corning; ThermoFisher) and adhered overnight prior to treatment with C-PAC [50 and 75μg/mL] as previously described^{17 (link)} or vehicle control (0.1% ethanol) dissolved in DMEM with 10% fetal bovine serum (Thermo Fisher). Cell lysates were harvested at 0, 24 and 48hr post-treatment using lysis buffer (1% Triton X-100, 50mM HEPES, pH 7.4, 150mM NaCl, 1.5mM MgCl2, 1mM EGTA, 100mM NaF, 10mM sodium pyrophosphate, 1mM sodium orthovanadate, 10% glycerol) with cOmplete™ EDTA-free protease and PhosSTOP phosphatase inhibitors (Sigma-Aldrich) following published protocols^{18 (link)}. Immunoblotting was performed using commercially available antibodies from Cell Signaling Technology (Danvers, MA): GAPDH (#2118; 1:40,000) and Santa Cruz Biotechnology: GSTT2 (#514667; 1:500) and HSP60 (#13115; 1:1000). Images were captured via the ChemiDoc Molecular Imager and band quantification with ImageLab analysis software (both Bio-Rad). Expression values normalized to loading controls were determined by chemiluminescent immunodetection with fold-change from vehicle reported.

Western blotting was used to assess (i) p-eNOS at S1177 normalized to total eNOS, (ii) LC3-II:LC3-I, and (iii) p62, HSP60, m-aconitase, Tom20, and VDAC normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using standard procedures. Antibodies for p-eNOS S1177, eNOS (1:500; Cell Signaling Technologies, Boston, Massachusetts, USA), LC3, HSP60, VDAC, m-aconitase, Tom20 (1:1000; Santa Cruz Biotechnology Inc., Dallas, Texas, USA), and p62 (1:1000; Abnova, Taipei, Taiwan) were obtained commercially. Densitometry of the immunoblot bands was quantified using Odyssey 3.1 (Li-cor, Lincoln, Nebraska, USA) (Symons et al. 2009 (link); Zhang et al. 2009 (link), 2010 (link), 2012 (link)).